Method for large-scale synthesis of long-chain nucleic acid molecule

ABSTRACT

Systems and methods for synthesizing long-chain nucleic acids molecules are disclosed. The systems and methods described in this application use a Ligation-Purification-Amplification (“LPA”) technique. The LPA technique requires first producing nucleotide sequences with the length of at least 500 bp-1 kbp by assembling smaller oligonucleotide fragments (30 bp-200 bp). The assembled nucleotide sequences are then purified and amplified. This technology can be used to achieve parallel amplification of three or more nucleotide sequences at the same time. In embodiments of the invention, a solid-phase ligation-purification method is adopted, in which nucleic acid molecules are fixed on the surface of beads or other solid particles in order to rapidly purify products obtained from the ligation reaction.

CONTINUITY DATA

The present application claims priority to Chinese Patent ApplicationNo. 201110193635.4, filed Jul. 12, 2011, along with PCT/CN2012/000818,filed Jun. 13, 2013, the entire disclosures of which are incorporatedherein by reference.

SEQUENCE LISTING

The present application contains a Sequence Listing, shown in table formin the Detailed Description, which has been submitted electronically inASCII format and is hereby incorporated by reference in its entirety.Said ASCII copy, created on Jan. 24, 2014, is named BVS001US_SL.txt andis 26,426 bytes in size.

TECHNICAL FIELD

The present invention relates to systems and methods for thelarge-scale, low cost synthesis of long-chain nucleic acid molecules byassembling short-chain oligonucleotides.

BACKGROUND OF THE INVENTION

In recent years, developments in the field of synthetic biology haveallowed researchers to synthesize a number of intermediates withpotential applications in, for instance, the bio-energy orpharmaceuticals fields, by changing bacterial or cellular metabolicprocesses. For example, in 2006, scientists used engineered yeast tosynthesize artemisinin intermediates, increasing the yield to reach 115mg/mL (Ro, D. K. et al. Nature, 2006, 440: 940-943). In 2009, GeorgeChurch's group accelerated the evolution of E. coli to increase theyield of lycopene by fivefold, through introducing a set ofoligonucleotides to modify the bacteria's genome (Wang, H. et al.Nature, 2009, 460: 894-898).

Scientists have also begun synthesizing complete genomes for livingorganisms. In 2008, the genome of Mycoplasma Genitalium, measuring 582kb in length, was successfully synthesized. In 2010, scientistsassembled a modified genome of about one million base pairs, and used itto produce a self-replicating Mycoplasma Mycoides (Gibson, D, et al.Science, 2010, 329:52-56.

One key development which has prompted significant growth in the fieldof synthetic biology is the maturation of gene synthesis technology. Inrecent years, DNA microarray technology has given researchers theability to synthesize genes on a large scale. Indeed, at present, onemicroarray can synthesize millions of oligonucleotides. However, thelengths of these oligonucleotides range generally between 60 bp to 200bp. Assembling these oligonucleotides into genes with sufficient length(i.e., greater than 1 kb) is a current challenge to researchers,particularly in circumstances where thousands of long-chain genes areneeded.

Currently, there exist two related strategies for addressing theseproblems. The first is to continue to improve synthesis techniques anddevelop new approaches to increasing the length of each oligonucleotide.The second is to develop an effective method to assemble millions ofshort oligonucleotides into long-chain genes in parallel. However, thefirst strategy depends on, and is still awaiting, further breakthroughsin new technologies. And while there has been some development withregards to the second strategy, these established approaches still havecertain drawbacks. For instance, Church successfully synthesized 47genes (measuring 35 kb) by selectively amplifying and assembling a groupof microchip-synthesized oligonucleotides (Kosuri, S. et al. NatureBiotechnology, 2010, 28(12):1295-1299). Church accomplished this byassembling a group of amplified oligonucleotides into a long-chainnucleotide sequence using a DNA microchip. Jingdong Tian's group alsoachieved synthesis of 74 genes (measuring 30 kb) by dividing amicroarray into distinct physical units, and then performing parallelsynthesis, amplification and assembly (Quan, J. et al. NatureBiotechnology, 2011, 29(5):448-452. However, both methods are not onlyvery costly, but are also limited in synthetic throughput.

This present application describes simpler and more feasible systems andmethods for synthetically assembling thousands of oligonucleotides intomultiple nucleotide sequences in parallel.

SUMMARY OF THE INVENTION

In one aspect of the invention, a method for synthesizing long-chainnucleic acid molecules through assembling short-chain oligonucleotidesis disclosed, the method comprising: phosphorylating a set ofshort-chain oligonucleotides; modifying at least one of the short-chainoligonucleotides in the set with a first chemical group; annealing theset of short-chain oligonucleotides; ligating the set of short-chainoligonucleotides in the presence of a ligase, wherein the ligationproduces at least one double-stranded long-chain nucleotide sequence;immobilizing the at least one double-stranded long-chain nucleotidesequence on solid particles modified with a second chemical group,wherein the first chemical group and second chemical group have anaffinity for one another; purifying the at least one double-strandedlong-chain nucleotide sequence; and, after purification, amplifying theat least one double-stranded long-chain nucleotide sequence.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic view of a method for synthesizing multipleoligonucleotides in accordance with an embodiment of the invention.

FIG. 2 shows the results of the synthesis of a single 554 bp DNAmolecule in accordance with an embodiment of the invention.

FIG. 3 shows the results of the synthesis of a single 731 bp DNAmolecule in accordance with an embodiment of the invention.

FIG. 4 shows the results of the synthesis of a single 1026 bp DNAmolecule in accordance with an embodiment of the invention.

FIG. 5 shows the results of the synthesis of a single 554 bp DNAmolecule in the absence of interference oligonucleotides, in accordancewith an embodiment of the invention.

FIG. 6 shows the results of the synthesis of a single 731 bp DNAmolecule in the absence of interference oligonucleotides, in accordancewith an embodiment of the invention.

FIG. 7 shows the results of the synthesis of a single 1026 bp DNAmolecule in the absence of interference oligonucleotides, in accordancewith an embodiment of the invention.

FIG. 8 shows the results of the synthesis of 554 bp, 731 bp, or 1026 bpDNA molecules in the absence of interference oligonucleotides, inaccordance with an embodiment of the invention.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

The systems and methods described in this application aim to achievelarge-scale synthesis of long-chain nucleic acid molecule, that is,first synthesizing short-chain oligonucleotides, and then assemblingthese short-chain oligonucleotides into long-chain nucleotide sequences.

In this application, the terms “nucleotide sequence,” “oligonucleotide,”“oligomer” and/or “nucleic acid molecule” are discussed with referenceto ribonucleic acid (“RNA”), deoxyribonucleic acid (“DNA”) and/or theirderivatives (including hybrids).

The systems and methods described in this application use aLigation-Purification-Amplification (“LPA”) technique. The LPA techniquerequires first producing nucleotide sequences with the length of atleast 500 bp-1 kbp by assembling smaller oligonucleotide fragments (30bp-200 bp). The assembled nucleotide sequences are then purified andamplified. This technology can be used to achieve parallel amplificationof three or more nucleotide sequences at the same time by use ofpolymerase chain reaction (“PCR”), if all the assembled nucleotidesequences are flanked with the same sequences. In embodiments of theinvention, a solid-phase ligation-purification method is adopted, inwhich nucleic acid molecules are fixed on the surface of beads or othersolid particles in order to rapidly purify products obtained from theligation reaction.

FIG. 1 shows a schematic view of a method of synthesizing multipleoligonucleotides in accordance with an embodiment of the invention. Thetechnology comprises three steps: ligation, purification andamplification, as shown in A, B, and C, respectively. In Step A(“Ligation”), a set of oligomers are mixed and treated withphosphorylation, then ligated, with the ends of two neighboring DNAfragments being joined together by the formation of phosphodiester bondsbetween the 3′-hyroxyl of one DNA termini with the 5′-phosphoryl ofanother. In this example, to facilitate the purification of the ligatedproducts, one of oligonucleotides was modified with biotin at the 5′end. These short-chain oligomers are subjected to annealing, and thenligation in the presence of a ligase, and finally form double-strandednucleotide sequences. In Step B, magnetic beads modified withstreptavidin are added to remove the substrates, incompletely connectedproducts or mismatched products. Finally, in Step C, the purifiedproducts are amplified by use of PCR or other known amplificationmethods.

The term “amplification methods” may refer to any method in whicholigonucleotides are used as templates and amplified in a linear ornon-linear manner, including but not limited to PCR, transcription,isothermal amplification, and the like. PCR, also known as in vitro DNAamplification technique, includes three steps, denaturation, annealingand extension.

In embodiments of the invention, the short-chain nucleic acid moleculesare first designed and synthesized. The long-chain nucleotide sequencesto be assembled are divided into a group of double-stranded short-chainnucleic acid molecules. The long-chain nucleotide sequences are dividedat points in a double-strand nucleotide sequence shifted from each otherby at least between three and nine nucleotides. The more nucleotidesbetween the divisions, the smaller the probability of mismatch becomes,thus increasing the accuracy of synthesis. The space of the divisionpoints along one strand preferably have the same length, but isdependent on the length of a short-chain nucleic acid molecule that canbe synthesized by use of the current art. Since the methods forsynthesizing single strand short-chain nucleic acid molecules are wellknown in the art, the cost is very low. For example, as one millionoligonucleotides with 60 bp in length cost only a few of hundreds USdollars. That is, each base is less than one cent.

Long-chain nucleotide sequences are at least 2 times the length of theshort-chain oligonucleotides. A long-chain nucleotide sequence typicallycomprises at least 700-1000 nucleotides, and each short chain nucleicacid molecules typically between 100-150 nucleotides. Synthesizingoligomers with a length of no more than 200 nucleotides is known in theart, such as from Agilent, LC science, etc.

In embodiments of the invention, the short-chain oligonucleotide ismodified with a chemical group, or “purification oligonucleotide.” Inaddition, modified solid particles with an affinity for the modifiedpurification oligonucleotide may also be present. In embodiments of theinvention, for example, the purification oligonucleotide could bemodified with biotin while the solid particles are modified withstreptavidin. In an embodiment of the invention, the purificationoligonucleotide may be modified with amino group and solid particlesmodified with a carboxyl group. In one example, the purificationoligonucleotide could be modified with azide group and solid particlesmodified with an alkyne group (or vice versa).

The short-chain oligonucleotides with a chemical group will beimmobilized by the modified solid particles. The solid particles mayinclude, but are not limited to, magnetic materials, silicon, silicondioxide, ceramics, polymers, silica or glass. In fact, any known solidmaterial capable of having its surface chemically modified as describedherein can be used. “Immobilization” refers to a process in which thenucleic acid molecule is closely linked to the solid mass through acovalent bond between a nucleic acid molecule with a first chemicalgroup (e.g., amino-NH2) and a solid body with a second chemical group(such as a carboxyl-COOH). Alternatively, the nucleic acid molecule maybe closely linked to the solid mass by forming a non-covalent bond withhigh affinity between a nucleic acid molecule and a solid body (e.g.,such as a nucleic acid molecule modified with biotin molecules and asolid body modified with avidin or streptavidin).

The short-chain oligonucleotides are then assembled into long-chainnucleotide sequences. The single-strand short-chain oligonucleotide ismixed in solution to form double-stranded “sticky end” nucleic acidmolecules, since blunt-end ligation is much less efficient than stickyend ligation. These sticky-end double-stranded nucleic acid moleculesmay be ligated in the action of ligase to form a completedouble-stranded long-chain nucleotide sequence. In embodiments of theinvention, the ligase might be T4 DNA ligase or Taq ligase.

To purify the assembled long-chain nucleotide sequences, the long-chainnucleic acid molecules immobilized on the solid particles are incubatedwith the solid particles, and then rinsed with the buffer. Besides thesolid-particle based purification method mentioned above, the nucleicacid molecule can be purified by using conventional high performanceliquid chromatography (“HPLC”), polyacrylamide gel electrophoresis(“PAGE”), capillary electrophoresis or gel electrophoresis method, orany other method known in the art. In an embodiment of the invention,the purification step includes a transcription step in order to enhancethe accuracy of amplified products or to reduce costs, since onlyexpected ligation transcripts would be reverse-transcribed and amplifiedlater. That is, the additional transcription and reverse-transcriptionperformance can remove transcripts without a correct nucleotide sequenceat the 3′ terminal.

At the amplification step, the nucleic acid molecules immobilized onsolid particles can be amplified by use of a known amplification method,such as PCR.

In embodiments of the invention, the 5′ or 3′ terminals of the assembledlong-chain nucleic acid molecule can be removed by use of enzymes. Ifthe amplified long-chain nucleotide sequences comprise the extrasequences used for purification and amplification other than theexpected nucleotide sequence, these extra sequences could be removedthrough enzymatic digestion. If the amplified long-chain nucleotidesequences are the expected nucleotide sequences, this step may not benecessary.

The advantage of the present invention over the traditional methods liesin the purification of assembled nucleic acid molecules prior toamplification. If there are any mismatched pairs between DNA bases, theassembled long-chain nucleic acid molecules may contain randomlyassembled products, incompletely ligated products or mismatchedproducts. Performing the amplification step after the assembly step,without purification, means that many more by-products are produced.This not only wastes raw materials, but also greatly increases thedifficulty and cost of separation. However, current systems and methodsfor synthesizing long-chain oligonucleotides have traditionally beenimmediately amplified before purification, resulting in a small numberof expected long-chain nucleic acid molecules at a high cost.

In the present invention, synthesized long-chain nucleic acid moleculespurified prior to amplification results in a significant decrease in theunexpected nucleic acid molecules, including randomly assembledproducts, incompletely connected products or mismatched products. Thisincreases the yield of the expected long-chain nucleic acid molecules.

Up until now, although the synthesis of short chain nucleic acidmolecules (less than 200 nucleotides) is easy and low-cost, the cost toobtain longer nucleic acid molecules is dramatically higher. Researchershave developed different strategies to obtain a certain number of longgenes (such as Jindong Tian's group, which achieved 74 individual genesthrough physical separation, totally about 30 kb), but ultimately, thethroughput capability is still subjected to the complexity and costconstraints. Because the present invention requires purification priorto amplification, it is feasible to perform the ligation, purificationand amplification of hundreds of nucleotide sequences in parallel. Andmore importantly, the present invention does not increase cost andoperational complexity.

As shown in FIGS. 2-8, the expected ligation products can be obtainedeven by use of initial oligonucleotides in low concentration such as0.01 fmol/μL, which is critically important for the DNA oligonucleotidesobtained from DNA microarray. The amount of DNA oligonucleotidessynthesized in microarray is about 1 fmol per spot. Traditionally, tomake use of these oligonucleotides for ligation, the amplification ofthese DNA oligonucleotides prior to ligation has been necessary.

For instance, FIG. 2 shows the results of the synthesis of a single 554bp DNA molecule. A set of nucleic acid molecules with 25-59 nt in length(17), the linker sequences (1 and 2) and purification oligonucleotide(Table 1), were obtained and subjected to the LPA technique inaccordance with embodiments of the invention. A single 554 bp DNAmolecule was synthesized, which is confirmed by cloning and sequencing.Even if the initial amount of each nucleic acid molecule is as low as0.01 fmol, the expected product can still be synthesized.

FIG. 3 shows the results of the synthesis of a single 731 bp DNAmolecule. A set of nucleic acid molecules with 25-60 nt in length (23),the linker sequences (1 and 2) and purification oligonucleotide (Table2), were obtained and subjected to the LPA technique in accordance withembodiments of the invention. A single 731 bp DNA molecule wassynthesized, which is confirmed by cloning and sequencing. Even if theinitial amount of each nucleic acid molecule is as low as 0.01 fmol, theexpected product can still be synthesized.

FIG. 4 shows the results of the synthesis of a single 1026 bp DNAmolecule. A set of nucleic acid molecules with 25-60 nt in length (33),the linker sequences (1 and 2) and purification oligonucleotide (Table3), were obtained and subjected to the LPA technique in accordance withembodiments of the invention. A single 1026 bp DNA molecule wassynthesized, confirmed by cloning and sequencing.

FIG. 5 shows the results of the synthesis of a single 554 bp DNAmolecule in absence of interference oligonucleotides. A set of nucleicacid molecules with 25-59 nt in length (17), the linker sequences (1 and2), purification oligonucleotide (Table 1) and a set of interferenceoligonucleotides (12) (Table 4), were obtained and subjected to the LPAtechnique in accordance with embodiments of the invention. A single 554bp DNA molecule was synthesized, confirmed by cloning and sequencing.

FIG. 6 shows the results of the synthesis of a single 731 bp DNAmolecule in absence of interference oligonucleotides. A set of nucleicacid molecules with 25-59 nt in length (23), the linker sequences (1 and2), purification oligonucleotide (Table 2) and a set of interferenceoligonucleotides (6) (Table 5), were obtained and subjected to the LPAtechnique in accordance with embodiments of the invention. A single 731bp DNA molecule was synthesized, confirmed by cloning and sequencing.

FIG. 7 shows the results of the synthesis of a single 1026 bp DNAmolecule in absence of interference oligonucleotides. A set of nucleicacid molecules with 25-60 nt in length (33), the linker sequences (1 and2), purification oligonucleotide (Table 3) and a set of interferenceoligonucleotides (6) (Table 3), were obtained and subjected to the LPAtechnique in accordance with embodiments of the invention. A single 1026bp DNA molecule was synthesized, confirmed by cloning and sequencing.

FIG. 8 shows the results of the synthesis of 554 bp, 731 bp, or 1026 bpDNA molecules in absence of interference oligonucleotides. A set ofnucleic acid molecules with 25-60 nt in length (17+23+33=73), the linkersequences (1 and 2), purification oligonucleotide (1) and a set ofinterference oligonucleotides (12+6+6=24) (Tables 1-6), were obtainedand subjected to the LPA technique in accordance with embodiments of theinvention. Three DNA molecules, including 554 bp, 731 bp, or 1026 bp,were obtained, respectively, confirmed by cloning and sequencing.

The results in FIGS. 5-8 indicate that the method described in thepresent invention demonstrates particularly strong anti-interferencecapability. As shown Tables 4-6, a number of interference DNAoligonucleotides were added in these experiments. In each case,interference DNA oligonucleotides contained the same sequence as one ofoligonucleotides to be assembled with the exception of 5 randomnucleotides (N represents any of adenine, thymine, guanine or cytosine),which could generate significant interference to the complementarypairing between short-chain DNA oligonucleotides. Even so, the expectedgenes were successfully synthesized. Thus these results indicate thatthe systems and methods described herein have very stronganti-interference capability and are suitable for large-scale, highaccuracy synthesis of long-chain nucleic acid molecules.

It will be readily understood by those persons skilled in the art thatthe present invention is susceptible to broad utility and application.Many embodiments and adaptations of the present invention other thanthose herein described, as well as many variations, modifications andequivalent arrangements, will be apparent from or reasonably suggestedby the present invention and foregoing description thereof, withoutdeparting from the substance or scope of the invention.

Accordingly, while the present invention has been described here indetail in relation to its exemplary embodiments, it is to be understoodthat this disclosure is only illustrative and exemplary of the presentinvention and is made to provide an enabling disclosure of theinvention. Accordingly, the foregoing disclosure is not intended to beconstrued or to limit the present invention or otherwise to exclude anyother such embodiments, adaptations, variations, modifications andequivalent arrangements.

Examples of embodiments of the invention are described below.

Example A

A set of DNA oligonucleotides are dissolved in water solution, mixed anddiluted to the concentration of 100 fmol/μl. These oligonucleotides aresubjected to phosphorylation by using T4 kinase (Polynucleotide kinase,New England Biolabs (NEB), LTD), then incubated at 37° C. for 1 hour.After phosphorylation, all DNA oligonucleotides are further diluted to aconcentration of each 1 fmol. Meanwhile, T1 beads (e.g., Dynal) aremixed with a purification oligonucleotide modified with biotin in 5′end, and incubated at room temperature for 15 minutes, then washed threetimes with buffer.

The phosphorylated DNA oligonucleotides are incubated with T1 beadsimmobilized with purification oligonucleotide at 65° C. for 2 hours. Inorder to ensure adequate hybridization between DNA oligonucleotides, thereactor is placed on top of a 220 rev/min shaker. The solution is slowlycooled to room temperature, and the beads washed to remove excess DNAoligonucleotides. Finally, T4 or Taq ligase was added into the solution,and the ligation reaction occurs overnight.

The ligated DNA products are then subjected to amplification throughPCR, then the amplified products are purified, cloned and sequenced.

Example B

A set of nucleic acid molecules with 25-59 nt in length (17), the linkersequences (1 and 2) and purification oligonucleotide (Table 1), werefirst mixed and then subjected to phosphorylation. After annealing, acomplete double stranded nucleic acid molecule can be formed in thepresence of T4 ligase. Subsequently, the magnetic beads modified withstreptavidin are used to purify the ligated products, which caneffectively remove the reaction substrate, incompletely connectedproducts or mismatched products. Finally, the purified products areenriched by use of nucleic acid amplification methods, such as PCR.

Example C

The magnetic beads are incubated with the purified oligonucleotide for15 min, then washed three times with buffer. Then the beads mixed withother DNA oligonucleotides, and are treated following the sameexperimental procedure, such as ligation, purification and amplificationdescribed in Example B.

Example D

Following the same experimental procedure described in Example B, onlythe magnetic beads are changed to glass particles, polymer particles,such as poly (meth) acrylamide, poly lactic acid (PLA), polylacticacid-glycolic acid polymer (“PLGA”), polyacrylic acid (“PAA”),polymethacrylic acid, with 2-hydroxyethyl(meth)acrylate,poly-N-isopropyl (meth) acrylamide, vinyl acetate or polypropylene, orpolyethylene amine.

Example E

A set of nucleic acid molecules with 25-59 nt in length (17), the linkersequences (1 and 2) and purification oligonucleotide (Table 1), arefirst mixed and then subjected to phosphorylation. After annealing, acomplete double-stranded nucleic acid molecule can be formed in theaction of T4 ligase. Subsequently, the product was purified by HPLC,which can effectively remove the reaction substrate, incompletelyconnected products or mismatched products. Finally, the purifiedproducts could be enriched by use of nucleic acid amplification methods,such as PCR.

Example F

Following the same experimental procedure described in Example B, onlythe HPLC purification was changed to use PAGE gel, gel electrophoresis,capillary electrophoresis or other purification methods.

Example G

Following the same experimental procedure described in Example B, exceptthat a set of nucleic acid molecules with 25-59 nt in length (17), thelinker sequences (1 and 2) and purification oligonucleotide (Table 1),12 interference oligonucleotides (Table 5) are added. The purifiedproducts were subjected to cloning and sequencing.

Example H

Following the same experimental procedure described in Example G, onlythe initial nucleic acid molecules includes a set of nucleic acidmolecules with 25-59 nt in length (totally 73), the linker sequences (1and 2), purification oligonucleotide (1) and a set of interferencesequences (totally 24) (Tables 1-6). The purified products weresubjected to cloning and sequencing.

In the examples for which the sequence listings are attached herein, thefollowing PCR primers were used: TCGAGCGGCCGCCCGGGCAGGT (as forwardprimer) (SEQ ID NO: 1); AGCGTGGTCGCGGCCGAGGT (as reverse primer) (SEQ IDNO: 2).

The invention claimed is:
 1. A method for synthesizing long-chainnucleic acid molecules through assembling short-chain oligonucleotides,the method comprising: phosphorylating a set of short-chainoligonucleotides; modifying at least one of the short-chainoligonucleotides in the set with a first chemical group; annealing theset of short-chain oligonucleotides; ligating the set of short-chainoligonucleotides in the presence of a ligase, wherein the ligationproduces at least one double-stranded long-chain nucleotide sequence;immobilizing the at least one double-stranded long-chain nucleotidesequence on solid particles modified with a second chemical group,wherein the first chemical group and second chemical group have anaffinity for one another; purifying the at least one double-strandedlong-chain nucleotide sequence, said purifying step including atranscription step followed by a subsequent reverse-transcription step;after purification, amplifying the at least one double-strandedlong-chain nucleotide sequence.
 2. The method of claim 1, furthercomprising the step of removing the terminal end of the at least onedouble-stranded long-chain nucleotide sequence through use of an enzyme.3. The method of claim 1, wherein the at least one double-strandedlong-chain nucleotide sequence is the entire nucleotide sequence to besynthesized.
 4. The method of claim 1, wherein the at least onedouble-stranded long-chain nucleotide sequence is comprised of thenucleotide sequence to be synthesized as well as two flanking sequencescomprising at least one PCR primer sequence and one oligonucleotide. 5.The method of claim 1, wherein the purification step is performed againafter amplification.
 6. The method of claim 1, wherein the solidparticles comprise at least one of the following: silicon, silicondioxide, ceramics, polymers, silica or glass.
 7. The method of claim 1,wherein the first chemical group comprises with biotin and the secondchemical group comprises streptavidin.
 8. The method of claim 1, whereinthe first chemical group comprises an amino group and the secondchemical group comprises a carboxyl group.
 9. The method of claim 1,wherein the first chemical group comprises an azide group and the secondchemical group comprises an alkyne group.
 10. The method of claim 1,wherein the ligase comprises at least one of the following: a T4 DNAligase or Taq ligase, a derivative of T4 DNA ligase, or a derivative ofTaq ligase.
 11. The method of claim 1, further comprising purifying theassembled nucleic acid molecules by at least one of the followingmethods: HPLC, PAGE, capillary electrophoresis or gel electrophoresis.